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. 2013 Feb 28;9(2):e1003319. doi: 10.1371/journal.pgen.1003319

Figure 2. Mitotic CDK promotes spindle elongation through FEAR pathway.

Figure 2

A. FEAR and cdc14-1 mutants suppress the growth defects in swe1Δ cells overexpressing CLB2. A control vector or a PGALCLB2 plasmid was transformed into WT, swe1Δ, swe1Δ net1-6Cdk, and swe1Δ spo12Δ cells. The growth of the transformants on 30°C glucose and galactose plates was examined as described in Figure 1A. The growth of WT, swe1Δ, swe1Δ cdc14-1 with a vector control of PGALCLB2 plasmid on glucose and galactose plates at 25°C is also shown. B. FEAR mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. Cells with the indicated genotypes were grown to mid-log phase in raffinose medium and then shifted to galactose medium. The cells were collected after 4 hr incubation at 30°C and fixed for DAPI staining. The percentage of binucleate cells is shown. The experiments were repeated for 3 times and at least 100 cells were counted for each sample. C. cdc14-1 mutants suppress the binucleate phenotype in swe1Δ cells overexpressing CLB2. WT, swe1Δ and cdc14-1 swe1Δ cells with a control vector or a PGALCLB2 plasmid were grown to mid-log phase in raffinose medium and then switched to galactose medium. After 4 hr incubation at 37°C, the cells were fixed for DAPI staining. The percentage of binucleate cells from three independent experiments is shown (n>100). D. FEAR mutants suppress the premature spindle elongation in swe1Δ cells overexpressing CLB2. cdc15-2 swe1Δ and cdc15-2 swe1Δ net1-6Cdk cells with a control vector or a PGALCLB2 plasmid were arrested in G1 phase in raffinose medium at 25°C and then released into galactose medium at 37°C. The budding index and the percentage of cells with an elongated spindle are shown in the left panels (n>100). The spindle morphology after 120 min release from G1 is shown in the right panel. The scale bar is 5 µm. E. Overexpression of CLB2 in swe1Δ mutant results in premature Cdc14 release. G1-arrested CDC14-5GFP, swe1Δ CDC14-5GFP and swe1Δ net1-6Cdk CDC14-5GFP cells with a vector or a PGALCLB2 plasmid were released into 30°C galactose medium containing 20 µg/ml nocodazole (NOC). Cells were collected over time and fixed to examine Cdc14 localization. The Cdc14 localization in cells at 3 hr time point is shown in the left panel. The percentage of cells with released Cdc14 was counted over time (right panel, n>100). Scale bar, 5 µm.